The kallikreins (E.C. 3.4.21.8) are members of the family of pancreatic serine proteinases that includes trypsin, chymotrypsin, elastase, urokinase and plasmin. Kallikrein is capable of hydrolysing kininogen, having the partial amino acid sequence ##STR1## to yield kallidin, a decapetide with physiological properties similar to bradykinin. [Han, Y.N. et al. FEBS Lett. 71, 45 (1976); Fiedler, F. et al. Adv. Exp. Med. Biol. 120A, 261 (1978)]. Bradykinin is believed to act as a smooth muscle hypotensive agent.
______________________________________ Table of Abbreviations ______________________________________ Pro = L-Proline Phe = L-Phenylalanine Arg = L-Arginine Val = L-Valine Cpc = Cyclopentane carbonyl Leu = L-Leucine Pec = 2-phenyl ethane carbonyl Cbo = Benzyloxy carbonyl Bz = Benzoyl &lt;glu = L-pyroglu HPK = Human plasma kallikrein HUK = Human urinary kallikrein Ser = L-Serine Met = L-Methionine Lys = L-Lysine Gly = Glycine BSA = bovine serum albumin pNA = para-nitro anilide ______________________________________
The present invention encompasses new compounds that act as unexpectedly potent substrates for plasma kallikreins. Sometime ago, applicants began a program to develop an assay for the kallikreins that combined the specificity of the chromogenic assay of Amundsen, E. et al. (in Pisano, J. J. et al. (eds.) Chemistry and Biology of the Kallikrein --Kinin System in Health and Disease, U.S. Government Printing Office 1976 p. 215) with the sensitivity of the radioassay of Beaven et al. (Clin. Chim. Acta, 32, 67 (1971). It had been shown that D--Pro--Phe--Arg--pNA is more reactive with plasma kallikrein than is Pro--Phe--Arg--pNA [Claeson, G. et al Haemostasis 7, 62 (1978)]. Applicants discovered that D--Pro--Phe--Arg--[.sup.3 H]--benzylamide is less reactive with plasma kallikrein than is Pro--Phe--Arg--[.sup.3 H]--benzylamide, indicating that it is not possible to predict the effects of changing leaving groups.
Applicants then began a search for amino acid residues (and related acyl groups) that might be used to replace Pro or (D)Pro in order to enhance the affinity of substrate for plasma kallikrein. Of twelve acyl groups thus surveyed, &lt;Glu (5--keto--L--Pro) proved to be the best. &lt;Glu--Phe--Arg--[.sup.3 H]benzylamide is highly reactive with human plasma kallikrein. The results of applicants indicate that subtle changes in the side-chain of the P.sub.3 (or proline) subsite of tripeptide substrates for the kallikreins can confer profound changes in selectivity and kinetic behavior.
Because of the role of kallikrein in regulation of blood pressure and renal blood flow, determination of kallikrein levels, particularly levels of active enzyme by hydrolysis of a substrate, is clearly relevant to clinical evaluation of disease. An assay for plasma kallikrein would be useful for screening patients for asymptomatic clotting defects, [Saito, H. et al. New Engl. J. Med. 305, 910 (1981)].
A variety of U.S. patents described substrates for proteolytic enzymes, some related to the kallikreins. See inter alia, U.S. Pat. Nos. 3,884,896; 4,016,042; 4,115,374; 4,137,225; 4,214,049; 4,215,047; 4,234,477; 4,242,329. See also American Hospital Supply PCT W080/00351, and applicants' pending application, U.S. Ser. No. 249,645 filed Mar. 31, 1981 which is a continuation of their application Ser. No. 34930 filed May 1, 1979. None of these references discloses the compounds of this invention as compositions of matter nor as substrates for plasma kallikrein. They do not describe the method of this invention for assaying plasma kallikrein.
For example, U.S. Pat. No. 3,886,136 discloses several chromogenic substrates for assaying enzymes of the class E.C. 3.4.4 (now class E.C. 3.4.21). Examples of this class include trypsin (E.C. 3.4.31.4), chymotrypsin (E.C. 3.4.31.1), plasmin (E.C. 3.4.21.7) and thrombin (E.C. 3.4.21.5), among others. The substrates disclosed in this patent have the general formula: ##STR2## wherein R.sub.3 and R.sub.4 are alkyl groups having 3-8 carbons, R.sub.4 can also be benzyl or phenyl, R.sub.5 is hydrogen or ##STR3## n is 2, 3 or 4, --NH--R.sub.6 is the chromogenic group, X is CH.sub.2 or a single bond, and R.sub.1 and R.sub.2 can be selected from a variety of groups which is not critical to this discussion. U.S. Pat. No. 4,016,042 discloses chromogenic or fluorogenic substrates for proteolytic enzymes of the class E.C. 3.4.31. These substrates are derivatives of Pro--X--Y--R where X is Phe, Tyr, phenylglycine or .beta.-cyclohexylalanine, Y is Arg or Lys and R is the chromogenic or fluorogenic group.
U.S. Pat. No. 4,137,225 discloses chromogenic substrates for proteases (serine proteases) of the class E.C. 3.4.21. This patent discloses substrates of the formula (D)A.sub.1 --A.sub.2 --A.sub.3 --R where A.sub.1 and A.sub.2 are selected from the group of amino acids Gly, Ala, Val, Leu, Ile, Pip, Pro or Aze, A.sub.2 can also be Phe, A.sub.3 is Arg, Lys or Orn and R is the chromogenic group.
Other U.S. patents which disclose chromogenic or fluorogenic peptide substrates include the following: U.S. Pat. No. 3,144,484 for trypsin (E.C. 3.4.31.4); U.S. Pat. No. 3,536,588 for Leu aminopeptidase (E.C. 3.4.11.1); U.S. Pat. No. 3,591,459 for amino acid arylamidase; U.S. Pat. No. 3,607,859 for neutral protease (microbial metalloenzymes, E.C. 3.4.23.4 ); U.S. Pat. Nos. 3,703,441; 3,769,173; 3,773,626; 3,892,631 and 4,177,109 for .gamma.-Glu transpeptidase (E.C. 2.3.2.2); U.S. Pat. No. 3,745,212 for pancreatic endopeptidases; U.S. Pat. Nos. 3,884,896, 4,191,808 and 4,191,809 for peptide peptidohydrolases such as class E.C. 3.4.21; U.S. Pat. No. 4,046,633 for renin (E.C. 3.4.99.19); U.S. Pat. Nos. 4,108,726 and 4,115,374 for angiotensin converting enzyme (peptidyldipeptide hydrolase, E.C. 3.4.15.1); U.S. Pat. No. 4,116,774 for Leu aminopeptidase, Cys aminopeptidase (1E.C. 3.4.11.3) and .gamma.-Glu transpeptidase; U.S. Pat. No. 4,138,394 for collagenase (E.C. 3.4.24.3); and U.S. Pat. No. 4,207,232 for factor Xa (E.C. 3.4.21.6).
The present invention encompasses novel derivatives of &lt;Glu--Phe--Arg--Y as new compositions of matter, wherein the indicator Y is either a radioactively tagged group, or a tag with fluorogenic, chromogenic or chemiluminescent properties. The compounds are unexpectedly effective substrates for plasma kallikrein. In addition, the method of assaying plasma kallikrein in this invention is novel.